IC4R010-Proteomic-2004-14730683

Project Title

• Proteome analysis of cultivar-specific deregulations of Oryza sativa indica and O. sativa japonica cellular suspensions undergoing Rice yellow mottle virus infection

Protein Extraction and 2-D PAGE

• Aliquots (1 g) of synchronized cell samples were ground to a fine powder in liquid nitrogen. Protein extraction and precipitation was performed in 10% v/v TCA in acetone with 0.07% v/v b-mercaptoethanol at 2207C for 1 h, followed by centrifugation for 10 min at 47C, 50 0006g. The pellets were washed twice with cold acetone containing 0.07% b-mercaptoethanol and centrifuged for 10 min at 47C, 50 0006g, and the pellets dried. Proteins were solubilized in 500 mL of lysis buffer (9 M urea, 4% w/v CHAPS,0.5% v/v Triton X-100, 0.5% IPG buffer, 3 mM tributylphosphine (TBP)). Finally, the sample was centrifuged 10 min at 207C, 60 0006g. Concentration of the supernatant was determined using protein assay (2D-Quant kit; Amersham, UK).

• At each time point, 2-DE was performed for two replications of both cultivars. All experiments of each replication were performed at the same time. Cell proteins (1 mg per sample) were loaded onto a IPG gel strip (240 mm, pH 4–7; Amersham). Isoelectric focusing (IEF) was conducted by using IPGphor (Amersham) system. IPG strips were rehydrated for 9 h prior to electrophoresis. IEF conditions were: 50 V for 10 h, 300 V for 10 min, 300–8000 V gradient for 3 h, and 8000 V for 13 h. The IPG gel strips were placed for 20 min in 15 mL of equilibration buffer (6 Murea, 0.375 M Tris-HCl, 30% v/v glycerol, 2% SDS w/v, 3 mM TBP). The second dimension was run on vertical 12% polyacrylamide-SDS gels. Gels were stained with colloidal Coomassie Brilliant Blue. The gels were scanned and spot intensities were analyzed using the software Image Master-2D (Amersham). A Student’s t-test was performed to evaluate the level of significance of any quantitative change in the level of analyzed proteins between control and each replicate at each time-point after RYMV inoculation. A change in protein abundance was considered significant if the difference between control and any time-point achieved the p , 0.05 significance level (t-test).

Labs working on this Project

• IRD, Institut de Recherche pour le Développement,Montpellier, France
• Laboratoire de Spectrométrie de Masse Bio-Organique,Strasbourg, France

Corresponding Author

• Marjolaine Ventelon-Debout:ventelon@mpl.ird.fr & Christophe Brugidou:brugidou@mpl.ird.fr